Fig. 3.
Fig. 3. Cytochemical staining of AChE activity in human megakaryoblasts is suppressed with maturation. (A) AChE cytochemical staining is reduced in PMA- more than in TPO-treated cells. Top: DAMI cells treated for 3 days with or without PMA or TPO were stained for AChE activity. A nuclear-association pattern of the cytochemical staining in the presence of 1 × 10−4 mol/L of the BuChE inhibitor (IO) was observed in all cells with or without PMA and TPO. Note the prominent decrease in color intensity within PMA-treated cells as compared with control and the moderate decrease in TPO cultures. Bottom (left panel): Untreated DAMI cells were subjected to AChE staining in the presence of 1 × 10−5 mol/L of the AChE inhibitor BW284C57 (BW); (right panel): BM cells derived from ALL patients were subjected to AChE staining without specific inhibitors. Cells in both bottom panels are unstained, reflecting the specificity of both the staining procedure on DAMI megakaryoblasts and the lineage-specific expression of AChE in megakaryoblastic, but not lymphoid cells. Size bar = 10 μm. Shown are representative pictures of one experiment of five in each panel. (B) PMA treatment increases cell areas (an analysis of the cells shown in Fig 3A, top panels). Percent of each cell population out of total cells analyzed as classified by their area is presented. Cell areas were grouped at 20 μm2 intervals and each point represents the largest area measured within that interval. Average cell areas expressed as mean μm2 ± standard deviation (SD) were: control cells (n = 360) 79.7 ± 45.4; PMA-treated cells (n = 189) 153.56 ± 87.6 (significantly larger than control cells, P < .0005), and TPO-treated cells (n = 281) 86.9 ± 54.5 (P < .1). Inset: Decreased AChE stained areas in PMA- and TPO-treated cells. Intracellular areas stained above threshold level were assessed as fractions of total cell areas using the INSITARE software (Applied Imaging, Dukesway, UK). The relative stained area for each DAMI cells preparation is presented in percentages as mean ± SD for each of the noted cell populations. ** P < .0005 as compared with control cells. (C) Flow cytometry analysis of DAMI cells treated for 3 days with PMA or TPO or without treatment (C) was performed by propidium iodide staining. Signals reflecting nuclei in each subclass of cells are presented as numbers (bottom) or in percentages of total cells (top). The first major peak represents the fraction of cells with a normal 2N complement of DNA. Subsequent peaks represent the proportion of cells with ploidies of 4N, 8N, 16N, and greater levels of DNA as shown on a log scale of the DNA contents and detailed in the above columns. Note that the peak distribution corresponds to that of the measured cell areas in Fig 3B. The results represent one experiment of more than 10 performed.

Cytochemical staining of AChE activity in human megakaryoblasts is suppressed with maturation. (A) AChE cytochemical staining is reduced in PMA- more than in TPO-treated cells. Top: DAMI cells treated for 3 days with or without PMA or TPO were stained for AChE activity. A nuclear-association pattern of the cytochemical staining in the presence of 1 × 10−4 mol/L of the BuChE inhibitor (IO) was observed in all cells with or without PMA and TPO. Note the prominent decrease in color intensity within PMA-treated cells as compared with control and the moderate decrease in TPO cultures. Bottom (left panel): Untreated DAMI cells were subjected to AChE staining in the presence of 1 × 10−5 mol/L of the AChE inhibitor BW284C57 (BW); (right panel): BM cells derived from ALL patients were subjected to AChE staining without specific inhibitors. Cells in both bottom panels are unstained, reflecting the specificity of both the staining procedure on DAMI megakaryoblasts and the lineage-specific expression of AChE in megakaryoblastic, but not lymphoid cells. Size bar = 10 μm. Shown are representative pictures of one experiment of five in each panel. (B) PMA treatment increases cell areas (an analysis of the cells shown in Fig 3A, top panels). Percent of each cell population out of total cells analyzed as classified by their area is presented. Cell areas were grouped at 20 μm2 intervals and each point represents the largest area measured within that interval. Average cell areas expressed as mean μm2 ± standard deviation (SD) were: control cells (n = 360) 79.7 ± 45.4; PMA-treated cells (n = 189) 153.56 ± 87.6 (significantly larger than control cells, P < .0005), and TPO-treated cells (n = 281) 86.9 ± 54.5 (P < .1). Inset: Decreased AChE stained areas in PMA- and TPO-treated cells. Intracellular areas stained above threshold level were assessed as fractions of total cell areas using the INSITARE software (Applied Imaging, Dukesway, UK). The relative stained area for each DAMI cells preparation is presented in percentages as mean ± SD for each of the noted cell populations. ** P < .0005 as compared with control cells. (C) Flow cytometry analysis of DAMI cells treated for 3 days with PMA or TPO or without treatment (C) was performed by propidium iodide staining. Signals reflecting nuclei in each subclass of cells are presented as numbers (bottom) or in percentages of total cells (top). The first major peak represents the fraction of cells with a normal 2N complement of DNA. Subsequent peaks represent the proportion of cells with ploidies of 4N, 8N, 16N, and greater levels of DNA as shown on a log scale of the DNA contents and detailed in the above columns. Note that the peak distribution corresponds to that of the measured cell areas in Fig 3B. The results represent one experiment of more than 10 performed.

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